A Heterogenous Population of Extracellular Vesicles Mobilize to the Alveoli Post-Injury

Abstract

Background:

Acute lung injury (ALI) and subsequent resolution following severe injury are coordinated by a complex lung microenvironment that includes extracellular vesicles (EVs). We hypothesized that there is a heterogenous population of EVs recruited to the alveoli post-injury and that we could identify specific immune-relevant mediators expressed on BAL EVs as candidate biomarkers of injury and injury resolution.

Methods:

Mice underwent 30% TBSA burn injury and BAL fluid was collected 4 hours post-injury and compared to sham. EVs were purified and single vesicle flow cytometry (vFC) was performed using fluorescent antibodies to quantify the expression of specific cell surface markers on individual EVs. Next, we evaluated human BAL specimens from injured patients to establish translational relevance of the mouse vFC analysis. Human BAL was collected from intubated patients following trauma or burn injury, EVs were purified, then subjected to vFC analysis.

Results:

A diverse population of EVs were mobilized to the alveoli after burn injury in mice. Quantitative BAL vFC identified significant increases in macrophage-derived CD44+ EVs (pre-10.8% vs. post-injury 13%, p<0.05) and decreases in IL-6 receptor alpha (CD126) EVs (pre-19.3% vs. post-injury 9.3%, p<0.05). BAL from injured patients also contained a heterogeneous population of EVs derived from myeloid cells, endothelium, and epithelium sources, with CD44+ EVs being highly detected.

Conclusion:

Injury causes mobilization of a heterogeneous population of EVs to the alveoli in both animal models and injured patients. Defining EV release after injury will be critical in identifying diagnostic and therapeutic targets to limit post-injury ALI.

Background:

Severe trauma and burn injury cause a systemic inflammatory response syndrome (SIRS) that can result in acute lung injury (ALI) and cause prolonged intensive care unit stays, complications, and death (1). ALI and acute respiratory distress syndrome (ARDS) remain significant clinical problems for patients who experience severe injury, with mortality rates approaching 31% (2). Dysregulation of the immune system as part of SIRS is a principal driver of lung inflammation (3). Understanding how inflammatory mediators in SIRS drive distant organ injury remains a key area of research, as effective pharmacotherapy options to prevent injury-induced ALI are lacking (4).

Extracellular vesicles (EVs) are cell-derived lipid-bilayer particles originally thought to serve as a waste disposal mechanism for cells but are now recognized as an important mechanism for transferring intracellular products between cells (5). EVs, which include exosomes and microvesicles, are formed through defined intracellular trafficking pathways, carry cargos, express canonical surface markers such as tetraspanins, and are released into the extracellular space(6). EV cargo can contain several biologically active products, including lipids, proteins, and nucleic acids that can transmit pro- and anti-inflammatory signals to local and distant sites (7). Given their role in intercellular communication, there is increased interest in EVs’ role in the pathogenesis of SIRS and lung inflammation (8, 9). Recently, EVs have been described as important mediators in the pathogenesis of numerous respiratory diseases, including asthma, transfusion-associated ALI, and sepsis-induced ALI (10, 11).

We have previously evaluated the effects of severe burn on the development of ALI, showing that a model of 30% total body surface areas (TBSA) burn injury is associated with histologic lung injury, increased lung permeability, increased pro-inflammatory mediator release, and increased mobilization of myeloid cells to the lung (12–16). Here, our goal was to define small EV mobilization to the alveoli post-injury as it may provide important insights for understanding the pathogenesis of ALI post-injury and could help identify potential diagnostic biomarkers or therapeutic targets. To this end, we analyzed bronchoalveolar lavage (BAL) fluid from a mouse model of severe burn injury and from human samples collected from severely injured patients to define the heterogeneity of EVs in the alveoli BAL post-injury.

Materials and methods:

Mouse Model

Male 6–8 week old C57BL/6J mice (#JAX000664) were housed with a 12hr light/dark cycle and provided food and water ad libitum. Prior to burn injury, mice were anesthetized with inhaled isoflurane and given a subcutaneous (SQ) injection of buprenorphine for analgesia. The dorsal fur of the mouse was clipped with an electronic hair clipper prior to a 7-second steam burn on the dorsal skin using a template that estimates 30% total body surface area burn that is associated with ALI as previously described (13, 16). Following injury, animals received fluid resuscitation with 1.5 ml of 0.9% normal saline SQ and were placed in their respective cages on a warming pad to recover. Sham mice underwent general anesthesia, fur clipping, injection of buprenorphine and normal saline but were not burned. Mice were selected randomly from their cage and allocated to their experimental group. No animals were excluded from analysis. All animal experiments were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee. Experimental details are reported using the Animal Research: Reporting of In Vivo Experiments guidelines (ARRIVE). A complete ARRIVE checklist was uploaded as supplemental digital content 1 (SDC1).

Collection of mouse bronchoalveolar lavage (BAL) fluid

Mice were first deeply anesthetized with isoflurane, followed by a tracheotomy for terminal collection of BAL fluid. Subsequently, 1 mL of 1x HBSS (Cat #88284, Thermo Fisher) with 100 μM EDTA (Cat #R1021, Thermo Fisher) buffer was gently injected into the trachea via a 27g needle on a syringe. BAL fluid was then recovered back into the syringe in a single aspiration (n=4 per group) and placed immediately on ice. Cellular components of the BAL fluid were removed via centrifugation at 3000 × g for 10 minutes at 4°C. Additional cellular debris were removed from the supernatant via a second centrifugation at 10,000 × g for 30 min. The remaining pellet was discarded, and the supernatant was stored at −80°C for future analysis.

Collection of human BAL samples

Human BAL samples (n=3) were collected after informed consent was obtained from a legally authorized representative. The Institutional Review Board approved the collection of BAL samples from intubated study subjects undergoing clinically indicated bronchoscopies, as decided by the patient’s primary surgical intensivist (IRB 180701). Using a clinical bronchoscope, BAL fluid was obtained by instilling 10 ml of normal saline into the bronchus under direct visualization. The fluid was suctioned into a sterile collection cup and placed immediately on ice. For initial processing, BAL fluid samples were centrifuged at 3,000 xg for 10 minutes at 4°C. A second centrifugation was performed at 10,000 xg for 30 minutes to further remove cellular debris. The resultant supernatant was then stored at −80°C for EV analysis.

Purification of BAL EVs

EVs from BAL fluid supernatant of mouse and human samples were isolated and collected by size exclusion chromatography using a qEV1 column (ICI-70, IZON science LTD, Portland, OR, USA) per manufacturer recommendations. EV-enriched 700 μL purified fractions were collected for further analysis.

Single vesicle flow cytometry (vFC) analysis

The primary outcome of this study was BAL EV surface marker analysis comparing sham and burn. EV samples (n=4 Sham vs. n=4 Burn) were analyzed using single vesicle flow cytometry (vFC) using the membrane dye vFRed (Cellarcus Biosciences Inc., La Jolla, CA, USA) in a kit format and processed per the manufacturer’s instructions (17, 18) and detected using a CytoFlex S flow cytometer (Beckman Coulter, Brea, CA). Briefly, all fractions were diluted with PBS to a 1:1000 dilution and incubated with a vFRed dye (#CBS4A, Cellarcus Biosciences Inc.) for one hour at room temperature to enable total EV quantification. Liposome (Lipo100, #CBS1, Cellarcus Biosciences Inc.) was used as a negative control, and mouse platelet-derived EVs (PLT, #CBS2, Cellarcus Biosciences Inc.) served as a positive control. vFRed signal and violet side scatter (V-SSC) were used to determine the vesicle concentration.

Expression levels of various proteins on the EV’s were determined via phycoerythrin (PE)-conjugated antibodies. The following proteins were evaluated in murine samples: CD44, CD126, CD274, and CD326. For human samples, the following proteins were evaluated: CD44, CD274, CD11b, CD31, CD16, CD105, Tetraspanin (TS) cocktail (CD9, CD63, CD81), CD11c, and cytokeratin. The following antibodies were used for the analysis of murine samples: CD44 (#130118–694, Miltenyi), CD126 (IL-6R) (#115806, Biolegend), CD274 (PD-L1) (#124308, Biolegend), and CD326 (#118205, Biolegend). The following antibodies were used for human sample analysis: CD44 (#163609, Biolegend), CD274 (#130-122-815, Miltenyi), CD11b (#130-110-611, Miltenyi), CD31 (#130-110-807, Miltenyi), CD16 (#130–113-955, Miltanyi), Tetraspanin (TS) cocktail containing CD9, CD63, CD81 (#CBS5-PE, Cellarcus Biosciences), CD105 (#130-112-321, Miltenyi), CD11c (#130-114-113, Miltenyi), and Cytokeratin (#130-112-932, Miltenyi). PE-conjugated antibodies were added simultaneously with vFRed dye in the same 1:1000 dilution as previously described and allowed to incubate for one hour at room temperature. 5 μL of 5 nM antibody was used for murine samples, and 5μl of 30 nM of antibody was used for human samples. All flow data were analyzed using FCS Express (Version 7, De Novo Software, Pasadena, CA).

Western blot

BAL EV samples were diluted to approximately 50,000 EVs/μL using NuPAGE^™ LDS Sample Buffer (#NP0008, Thermo Fisher). For protein reduction, 1 μL of fresh 1M DTT was added, and the sample was heated at 70°C for 10 minutes. All samples (n=3 per group) were then loaded onto a 1 mm pre-cast 12% Bis-Tris Mini Gel (#NP0342BOX, Thermo Fisher) and subjected to electrophoresis at 200 V for 42 minutes. Proteins were then transferred to a PVDF membrane (#LC2005, 0.45 μm, 8.3 × 7.3 cm, Thermo Fisher) via electrophoresis at 160 mV for 35 minutes. Memcode stain (#24585, Thermo Fisher) was then used to confirm protein loading. Each membrane was then blocked with 3% Nonfat Dry Milk (#9999, CST, MA, USA) in 1X Tris-buffered saline (#9997, CST) containing 0.05% Tween 20 for 1 h. Membranes were then incubated overnight at a dilution of 1:1000 with the primary antibodies anti-CD81 (#10037, CST) or anti-Alix (#92880, CST). After primary antibody incubation, membranes were incubated with secondary antibodies for 1 hour at room temperature. The following secondary antibodies were used: Anti-rabbit IgG, HRP-linked antibody (#7074, CST), and anti-mouse IgG, HRP-linked antibody (Cat# 7076, CST). The blots were imaged using SignalFireTM ECL Reagent (#12757, CST), and fluorescence was detected with a Xenogen IVIS-Lumina (Caliper life science Inc., Hopkinton, MA, USA). Band intensities were quantified using Living Image software (Ver.4.3.1, PerkinElmer, Waltham, MA).

Statistical analysis

Data were presented as a mean ± standard deviation. Group comparisons were performed using Student’s t-test for normally distributed values, and *p-values <0.05 were considered statistically significant. Statistical analyses were conducted using Prism 6.0 software (GraphPad Software, La Jolla, CA, USA).

Results:

Quantification of EV heterogeneity in BAL fluid of mice using single vesicle flow cytometry

To address the limitations of many bulk approaches for the analysis of EVs in biological fluids such as EVs in BAL, we performed a series of purification and analysis steps on BAL from mice with burn injury compared to sham mice. We have previously shown that this burn injury model leads to SIRS that affects the lungs within 4 hours (13, 16). Therefore, BAL was collected 4 hours post-injury from burn vs. sham mice, and EV separated from cells by serial centrifugation and the EVs purified by size exclusion chromatography (SEC, Fig. 1A). SEC facilitates the separation of EVs from free protein in fractions that are each subjected to quantification by staining with a fluorescent membrane dye, vFRED, that has been previously shown can be used for the enumeration of EVs (Fig. 1B). Western blotting of EV fraction three (F3) from each mouse group was subjected to immunoblotting, a bulk technique that demonstrated expression of canonical EV markers like CD81, which did not change between groups, and Alix, which was increased in the burn group vs. the sham group (Fig. 1C). The recovery and purification procedure yielded lung EVs that were similar in size between mouse BAL from the sham and burn groups (Fig. 1D). We measured the diameter of EVs from each fraction to demonstrate change in the distribution of EV size based on SEC fraction (Fig. 1E).

Figure 1. Characterization of BAL EVs.

EVs from murine BAL samples from both sham (=4) and burned mice (n=4) were purified with size exclusion chromatography (SEC) and characterized using known EV markers. (A) Schematic demonstrating how BAL EVs were collected and isolated using SEC. (B) Fractions three (F3) and four (F4) from SEC purification demonstrated the highest concentration of EV’s. (C) Western blot analysis showed that the EV markers Alix and CD81 were expressed on BAL EVs from sham and burn samples. (D) There is no difference in EVs size when comparing sham vs. burn BAL samples from F3. (E) Histograms demonstrating EV size for each SEC fraction.

In contrast to bulk techniques like western blotting, single vesicle flow cytometry (vFC) enables the analysis and quantification of individual EVs. Representative examples of EVs stained with vFRED, which enables the detection of EVs based on membrane fluorescence rather than typical light scatter for cells, were stained with fluorescently-labeled antibodies to the tetraspanins CD9, CD63, and CD81 (Fig. 2A). Negative controls include the assay of the sheath buffer and the absence of primary antibody on vFRED-stained EVs (Fig. 2A, left) along with isotype controls (data not shown). These negative controls established the gate for EVs stained with fluorescent antibodies. We found that the tetraspanins CD9, CD63, and CD81 were highly expressed (range of 46–83%) on EVs purified from BAL of both sham and burn group mice. These studies established the baseline characterization of EVs from sham mice and the first analysis of EV heterogeneity by vFC from a BAL sample in an injury model.

Figure 2.

EVs from murine burn BAL samples (n=4/group) have increased expression of CD44 and CD126 compared to sham samples. Expression of CD274 and CD326 were unchanged. Protein expression levels were measured by vFC. (A) vFC analysis showed that sham and burn BALF EVs express the tetraspanins CD9, CD63, and CD81. Burn EVs have increased expression of (B) CD44 and (C) CD126 compared to sham EVs. Burn EVs have unchanged expression of (D) CD274 and (E) CD326 compared to sham EVs, *p<0.05.

Alveolar EV populations change following severe burn injury

Based on the characterization of EVs from the sham and burn groups, we used vFC to analyze the expression of surface proteins that are highly relevant in intercellular signaling between immune cells and the airway epithelium. To this end, we analyzed the expression of CD44. In this hyaluronic acid receptor, we observed an increase in expression following burn injury (Fig. 2B). In contrast, the expression of CD126 (Fig. 2C), the interleukin 6 receptor (IL-6R) was decreased in burn injury compared to sham mice. Expression of CD274, programmed death-ligand 1 (PD-L1) (Fig. 2D), and CD326 (Fig. 2E), the epithelial cell adhesion molecule (EpCAM), were both unchanged. Together, these findings provide specific examples of signaling mediators relevant to immune-epithelial cross-talk that may mediate inflammation responses in the lung following severe injury.

A heterogeneous population of EVs is present in the BAL of severely injured patients

To identify EV surface proteins in a small patient sample cohort that could be used for larger studies and to guide studies in animal models, we enrolled ICU patients who were intubated as part of their standard of care (Fig. 3A). From a combination of trauma and burn patients, BAL was collected, subjected to serial centrifugation and SEC as described above. EVs were then subjected to staining with the membrane stain vFRED to gate the EV population (not shown), followed by staining with a cocktail of antibodies directed to CD9, CD63, and CD81 (Fig. 3B). In this representative sample, we identified 46% of EVs were positive for staining with these canonical EV markers. Although the EV field is highly focused on the use of these tetraspanins for validation studies, we next considered the potential of other protein targets that we and others have recently identified in mouse and human EV studies. We observed that molecules such as CD44 and CD274 were highly expressed, similar to mouse BAL EVs (Fig. 3C), while other proteins such as CD11b, CD31, and CD16 were all higher than the classic tetraspanins. A quantitative approach to the analysis of mouse and human BAL enables the cross-comparison of conserved EV markers relevant to ALI that exploit the advantages of animal models that have control groups and human samples that have clinical relevance.

Figure 3.

Injured human BALF EVs (n=3 patients) express known EV markers and multiple immune related marker proteins. (A) Enrollment table of human patients with relevant clinical information. (B) CD9, CD63, CD81 expression on injured human BALF EVs measured by vFC. Human samples express CD9, CD63, and CD81 (C) Expression levels of multiple immune related marker proteins on injured human BALF EVs measured by vFC. Human injured samples express multiple immune related markers, most notably CD44, CD274, and CD11b.

Discussion:

SIRS-induced ALI remains a significant clinical problem in patients admitted following severe injury, with no targeted therapies available to decrease its associated morbidity and mortality. Here, we sampled alveolar EVs in a murine model of injury and in human patients after trauma/burn. In mice, we identified a heterogeneous population of EVs in alveoli, finding that burn injury was associated with a significant increase in CD44⁺ EV’s while IL-6R⁺ (CD126) EVs were decreased post-burn injury. We obtained BAL from severely injured patients and confirmed the presence of EVs in the alveoli post-injury and identified CD44 and CD274 as highly expressed molecules. Identifying how EVs participate in immune-epithelial cross-talk and contribute to ALI and resolution of injury will be critical to the development of diagnostic and therapeutic targets in the future.

Circulating biomarkers associated with the development of ALI after trauma have been identified; however, none are used clinically for either diagnostic or therapeutic purposes and are specific to the SIRS response rather than to the pathogenesis of lung inflammation (19). Given the importance of the alveolar microenvironment in ALI, direct sampling is critical in determining the biological mechanisms of ALI pathogenesis. BAL fluid can be easily obtained through flexible bronchoscopy, a procedure widely used in clinical practice as a diagnostic tool for infectious and inflammatory pulmonary disease and allows for direct sampling of alveolar fluid (11).

We have demonstrated that alveolar EVs express tetraspanins and other cell surface markers from multiple cellular sources suggesting that these EVs may have a role in cross-talk during the lung inflammatory response. Based on the EV heterogeneity that we have demonstrated here, studying EVs in the lung requires high-resolution evaluation rather than bulk analysis using traditional techniques such as Western blotting. The International Society of Extracellular Vesicle consensus statement recommends reporting EVs based on their size as small (<200nm) vs. large (>200nm) EVs rather than attempting to characterize as exosomes or microvesicles as determining their specific biogenesis pathway is very difficult (20). We utilized SEC to concentrate small EVs as they have defined trafficking pathways and biological activity related to cell-cell communication. This SEC technique fractionates all EVs based on size and vFC shows that the relative abundance of exomeres and especially microvesicles is low. We utilized vFC to characterize EV size and expression of surface proteins on these small EVs. This rigor is required to consider the presence of EVs that may originate from different sources with unique biological activity, which may differentially contribute to the cross-talk between immune cells, the lung epithelium, and the pulmonary endothelium during homeostasis, lung inflammation, or injury resolution (21). Future studies will be needed to demonstrate the time course of EV expression in the alveoli post-injury and determine the biological activity of specific and immune-relevant EVs in the lung.

We found that EVs expressing CD44 increased in the early time points in a burn model that we have previously shown is associated with ALI (12, 13, 16). CD44 is a transmembrane adhesion molecule expressed on several cell types, including leukocytes. It contributes to the innate immune response to injury through monocyte proliferation, phagocytosis, and CD44 ligand hyaluronic acid clearance (22, 23). The importance of CD44 in the resolution of ALI has been shown in models of non-infectious lung injury, where CD44 knockout mice were found to have exaggerated lung inflammation, increased mobilization of neutrophils to the alveolar space, and increased mortality (23, 24). CD44 has been studied as a therapeutic target in cancer based on its effects on proliferation of cancer cells (25); however, additional studies are needed to determine if CD44 could be a useful biomarker and therapeutic target in post-traumatic ALI.

We also found that CD126, the IL-6R, decreased in the alveoli of mice post-burn injury. The pro-inflammatory cytokine IL-6 signals through the IL-6R and is a central mediator of the acute-phase response of immune cells. IL-6 can also signal through a soluble form of the IL-6R through a process known as trans-signaling (26). One mechanism by which soluble IL-6 is released is via EVs (27). Binding of IL-6 to the membrane-bound vs. soluble IL-6R is believed to be a dynamic regulatory mechanism to control pro- vs. anti-inflammatory signaling (28). Blockade of IL-6R has been considered as a potential therapeutic in inflammatory diseases such as inflammatory bowel disease (29). Therefore, targeting EVs expressing the IL-6R may be a target in treating SIRS-induced ALI.

To our knowledge, this is the first study to utilize vFC to evaluate surface markers on EVs in the BAL fluid of severely injured patients. A prior study obtained BAL to compare smokers versus non-smokers with COPD (30). In that study, the authors identified differences in macrophage and epithelial-derived EVs between smokers vs. non-smokers with COPD, while no differences were found in EVs collected from plasma. This further supports that utilizing BAL to obtain EVs from the alveolar space more likely represents the complexity of the lung inflammatory response compared to circulating markers. Another study collected BAL from patients following lung transplantation to characterize exosome markers to identify early biomarkers of allograft rejection (31).

There are limitations to the data presented in this study. The vFC analysis is limited to small EVs based on our use of SEC. While we believe this results in the analysis of EVs most relevant to SIRS-induced ALI, we have not analyzed large EVs or their potential to contribute to alveolar EV heterogeneity. The number of human BAL samples analyzed is small and was not planned as a comprehensive analysis of the human EV response to injury and was therefore not correlated to other inflammatory markers or outcomes. Instead, we wanted to determine whether there was any similarity between human alveolar EVs and EVs that we identified in our mouse studies. A main focus of our laboratory is defining human-relevant mediators of the inflammatory response, so it was critical to determine if EV source after injury in mice was relevant to human lung inflammation (16, 32). As alveolar EVs had yet to be characterized post-injury, to our knowledge, we felt there was value in confirming their presence in human alveoli and measuring trafficking of EVs based on their surface markers to gain insights on EV trafficking post-injury.

Here, we performed a baseline characterization of alveolar EVs in an animal model of burn injury and in severely injured patients to demonstrate EV heterogeneity using vFC. We identified signaling mediators relevant to immune-epithelial cross-talk in the lung that may mediate ALI due to SIRS. Defining EV release after injury will be critical in identifying diagnostic and therapeutic targets to limit post-injury ALI.